The study received approval from the local ethics and research committee of the Shanghai Ninth People’s Hospital (SH9H-2021-T357-2). In 2021, the Ninth People’s Hospital, affiliated with the Shanghai Jiaotong University School of Medicine, initiated the recruitment and subsequent enrollment of a three-generation, seven-member family presenting with VWS as well as unrelated control subjects. Before the commencement of any research-related activities, written informed consent was secured from all participants. For individuals under 18 years of age, informed consent was received from their respective guardians in strict adherence to the tenets of the Declaration of Helsinki. Physical exams were conducted by two surgeons to examine any possible organ malformations. The proband’s mother underwent a detailed review of her prenatal exposure history, encompassing factors such as smoking habits, alcohol consumption, medication and supplement intake, prior illnesses, and potential radiation exposure.

To pinpoint potential pathogenic gene mutations within the pedigree, we conducted WES of the genomic DNA isolated from the family members. Venous blood (2 mL per individual) was drawn from each participant and anticoagulated with EDTA. Subsequent genomic DNA isolation was facilitated using a Qiagen DNA extraction kit according to the manufacturer’s recommended protocol. Amplification of the genes of interest was performed by polymerase chain reaction (PCR) using primers encompassing all exonic and adjacent intronic regions (primer sequences available upon request). WES was carried out on a BGISEQ-500 platform (BGI, China) to ascertain the underlying mutation. The data from each sample underwent sequence alignment to the human reference genome (GRCh37/hg19) utilizing the Burrows-Wheeler Aligner tool (Oxford, UK). Mutation discernment was facilitated via the Genome Analysis Toolkit (GATK; accessible at https://www.broadinstitute.org/gatk/guide/best-practices). Metrics encompassing sequencing depth and coverage for each specimen were deduced from the alignment outputs. Annotations and pathogenicity predictions were subsequently orchestrated through SnpEff (detailed at http://snpeff.sourceforge.net/SnpEff_manual.html).

Primer3 (https://bioinfo.ut.ee/primer3/) was used to design primers for the identified pathogenic mutations. These primers were then added to the samples from the VWS patients and healthy individuals, and the target genes were amplified using PCR. The samples then underwent Sanger sequencing to compare the obtained results with the reference sequence (NM_006147.4).

The antibodies used in our study were procured from two main suppliers: Abmart and Cell Signaling Technology. From Abmart, we obtained the mouse monoclonal antibody targeting DYKDDDDK-Tag (3B9) (M20008S) for Western blot, an anti-EGFP antibody (PS09757S), Alexa Fluor 488-labelled goat anti-mouse IgG (M21011L), and Alexa Fluor 594-labelled goat anti-rabbit IgG (M21014L) for immunofluorescence. Cell Signaling Technology provided us with rabbit antibodies against SMAD3 (9523S), phospho-SMAD3 (9520S), phospho-SMAD2 (18338S), and GAPDH mouse antibodies (2118S) as well as HRP-linked anti-rabbit IgG (7074P2) and anti-mouse IgG (7076S) secondary antibodies.

The full-length IRF6 sequence (NCBI Refseq: NM_006147.4) was amplified using standard PCR techniques and subsequently inserted into a pCDH-SBP-HIS₈ vector. The mutations of IRF6 were crafted through PCR and then integrated into the pCDH-SBP-HIS₈ vector. Likewise, the full-length TGFβ2-AS1 gene (NCBI Refseq: NG_027721.3) was synthesized using basic PCR and then subcloned into the pGreen-GFP-puro vector.

Human oral keratinocytes (HOKs) isolated from human mucosa were purchased from ScienCell (Carlsbad, CA, United States) and maintained in Oral Keratinocyte Medium (ScienCell). The HOKs were inoculated into 10-cm Petri dishes containing Dulbecco Modified Eagle Medium (DMEM; Gibco, Carlsbad, CA, EUA) containing 4 mM l-glutamine, 1.5 g/L sodium bicarbonate and 4.5 g/L glucose and cultured at 37°C in a humidified atmosphere of 5% (v/v) CO2. The culture medium was supplemented with 10% fetal bovine serum (FBS), 100 μg/mL streptomycin, 100 μg/mL penicillin and 0.1% gentamicin. After culturing, the cells were washed twice with phosphate-buffered saline (PBS) to remove any impurities or residual media. The cells were then treated with 2 mL of 0.05% T/E solution and incubated for about 5 minutes until each cell morphed into a distinct spherical shape. This transformation is followed by centrifugation at 1,000 rpm for 3 minutes. The cells were then rejuvenated with fresh culture mediumand then transferred to a new dish and cultured at a density of 5,000 cells/cm², ensuring optimal space for growth and nutrient absorption.

One of the intriguing experiments involved the lentivirus-induced overexpression of IRF6 in HEK-293T cells. In this process, 4 ng of either wild-type IRF6 or mutated IRF6 pCDH-SBP-HIS₈ plasmid, combined with 1 ng each of pCMVdelta8.2 packaging vector and VSVP enveloped vector, were co-transfected into HEK-293T cells housed in 60 mm cell-culture dishes from NEST. Following a 48 and 72-h incubation period, the virus-laden supernatant was carefully harvested. The supernatant was collected by filtration through a 0.45 mm PES syringe filter (Thermo Fisher, United States) to ensure purity. The cells were then exposed to this lentivirus in the presence of 8 mg/mL polybrene (Sigma-Aldrich, St. Louis, MO, United States) to initiate the infection process. In the final phase of lentiviral infection, the HOK cells demonstrating positive signs of infection were cultured in 5 mg/mL puromycin (Solarbio) for 5 days This ensured that only the cells proficiently expressing the desired traits were selected.

After washing thrice with PBS, the HEK-293T cells overexpressing IRF6 were fixed with 4% paraformaldehyde for 10 min at room temperature, and the cell membranes were permeabilized with 0.2% Triton X-100 for 1 hour. Following incubation overnight at 4°C with the appropriate primary antibodies, the permeabilized cells were incubated with a goat anti-mouse FITC-conjugated secondary antibody for 1 hour at room temperature, after which the cells were stained with DAPI to visualize the cell nuclei. Aliquots of the cell mixtures were loaded onto microscope slides, which were supplemented with fluorescence decay-resistant medium (Beyotime) and mounted under a confocal laser-scanning microscope (Leica) for visualization.

Total RNA was extracted from a population of 1 × 10⁶ HOK cells using the TRIzol reagent (Invitrogen). The total RNA extracted from lentivirus-infected HOK cells constructed in the above steps was reverse transcribed into cDNA, and the cDNA was fragmented to construct the library. The QC-qualified libraries were sequenced (Illumina PE150), and clean data were filtered out after obtaining the raw data. Subsequently, the sequencing reads were aligned to the mouse reference genome (GRCm38/mm10) by HISAT2 software. The mRNA expression abundance was analysed using StringTie software, and differential expression analysis was performed on the transcript data of each group using DESeq2 R (version 1.10.1) software package. Gene set enrichment analysis (GSEA) and gene ontology (GO) enrichment analyses were performed on the screened differentially expressed genes to obtain the biological processes and signalling pathways that might be regulated by IRF6.

Biological variability was assessed by conducting principal component analysis (PCA) on the pre- and post-normalization data using the R package (version 3.6.1). Box plots using the ggplot2 package in R (version 3.6.1) indicated relatively uniform standardized data distribution. After standardization of the raw data, mRNAs of at least 5 out of 15 samples with current or edge markers were selected for further data analysis. p-values for assessing the differences in transcriptional levels were calculated using an unpaired t-test and then adjusted with the Benjamini–Hochberg FDR method. Differentially abundant mRNAs were then further filtered using an FC cutoff of >1.5 and an FDR cutoff of <0.05 to identify the DEGs. The volcano plot and heatmap of the DEGs were constructed using GraphPad Prism (version 8.0.1) and R/pheatmap package (version 1.0.12).

HOK cells were stably transfected with pCDH-SBP-HIS₈-IRF6-c.748C>T. The cells were lysed in NP-40 protein lysis buffer for 30 min at 4°C, and Western blotting was performed to quantify the expression levels of mutant IRF6. The SBP-His₈-tagged mutant IRF6 protein was precipitated by a two-step affinity purification over streptavidin-agarose resin and nickel resin columns. The precipitate containing the complex copurified with SBP-HIS₈-IRF6-c.748C>T:p.R250X was digested with sequencing-grade trypsin (Promega). The resulting peptides were analyzed by LC-MS/MS in ESI mode using an Eksigent 2D nanoLC coupled in-line with an LTQ-Orbitrap mass spectrometer. More detailed steps were conducted as described previously (Beaty et al., 2010).

HOK cells were collected, washed twice with precooled PBS, and lysed in NP-40 protein lysis buffer at 4°C for 30 min. The protein concentration was measured using a bicinchoninic acid (BCA) assay kit (Thermo Fisher Scientific). Immunoblotting was conducted as previously described (Cui et al., 2022). For immunoprecipitation, total cell lysates were incubated with specific antibodies for 2 hours followed by rotation with flag beads overnight at 4°C. The resulting beads were washed three times with NP-40 lysis buffer and mixed with 1x SDS loading buffer before being boiled for 10 min. Finally, the supernatant was analyzed by immunoblotting.

Total RNA was extracted from the HOK cells utilizing the TRIzol reagent (Thermo Fisher, 98597101) and reverse-transcribed into complementary DNA (cDNA) using the PrimeScript RT reagent kit with gDNA Eraser (YEASEN, H2107011). The quantitative PCR (qPCR) was performed on an ABI 7900 real-time PCR (Applied Biosystems) using the SYBR Premix Ex TaqTM II (Perfect Real Time, Takara, A8402-1); GAPDH served as an endogenous control to normalize gene expression levels. The raw data were analyzed. The 2^–ΔΔCT method was employed for accurate quantification of gene expression levels. Table 1 shows the sequences of the qPCR primers (Shanghai Sangon Biotechnology, Shanghai, China).

All data reported herein represent as the mean ± standard error of the mean (SEM). Statistical significance was assessed using Student's t-test. Significance levels are denoted as follows: *p < 0.05, **p < 0.01, and ***p < 0.001, with the latter indicating the most statistically significant difference.

This study entails a detailed examination of a consanguineous family pedigree of three generations. The proband III:1 had a clinical diagnosis of VWS (Figure 1A). This diagnosis was primarily attributed to distinct physical anomalies: a complete cleft lip and a lower lip pit, both prominently exhibited on the right side of the face (Figure 1B). The analysis of the family pedigree revealed intricate patterns of inheritance and phenotypic manifestations spanning multiple generations. In the second generation, the family comprised three members (Figure 1A). In the second generation of the family line, II:1, II:2, and II:4 all had the disease, and all presented with a lower lip pit. However, both II:1 and II:4 died in childhood from other diseases, and the phenotype of the patients could not be further characterized. This continued into the third generation, as the prototype III:1 also presented with a right cleft lip. (Figure 1B). A scrutiny of the family’s generational health portrait unveiled a discernible autosomal-dominant pattern of inheritance. This pattern underscored the familial passage of the condition, laying bare a roadmap of genetic transmission and phenotypic expression that holds the promise of unlocking deeper insights into the enigmatic nature of this condition.

In this comprehensive study, WES was performed on three patients afflicted with a specific genetic disorder and one unaffected individual (Table 2). A total of 90 mutated genes were uncovered through this process. We employed Sanger sequencing to authenticate the disease-associated nonsense mutation, which included a hitherto unreported mutation of IRF6 (c.748C>T:p.R250X) located in exon 7 (Figure 1C). This novel discovery revealed the stop-gain mutation situated within the SIMR/IAD-domain (Figure 1E). Notably, this mutation was not present in any of the unaffected family members and had not been previously cataloged in the dbSNP Database. A meticulous review of existing literature disclosed a significant association between diverse mutations within the IRF6 gene and the onset of both cleft lip/palate and VWS. Our in-depth analysis of protein structure and functional prediction underscored the profound impact of this specific mutation. It instigates the transformation of the arginine (Arg) codon at position 250 within the IRF6 gene into a stop codon, engendering a significant alteration in the resultant protein structure. In a comparative analysis spanning various species including Homo sapiens, Mus musculus, Rattus norvegicus, Cavia porcellus, Pan troglodytes, Equus caballus, Papio anubis, and Oryctolagus cuniculus, the Arg250 residue was found to be evolutionarily conserved (Figure 1D). The conservation of this residue across multiple species underscores its fundamental role, and mutations at this site could potentially have far-reaching implications for protein functionality and, by extension, the development and progression of associated genetic disorders. These findings lay a robust foundation for further research aimed at elucidating the intricate relationships between IRF6 mutations and their clinical manifestations, paving the way for enhanced diagnostic precision and the development of targeted therapeutic interventions.

HOK cells stably expressing Flag-tagged IRF6-c.748C>T:p.R250X or wild-type IRF6 were prepared by lentiviral infection and puromycin selection (Figure 2A). Immunofluorescence investigations revealed distinct localization patterns for the wild-type and mutated (c.748C>T:p.R250X) IRF6 in the HOK cells. Wild-type IRF6 was expressed in both the cytoplasm and nucleus, while the mutated IRF6 was predominantly localized to the cytoplasm (Figure 2B), indicating a potential impact on its nuclear import and functionality therein. We advanced our exploration to elucidate the disparity in protein interactions between the wild-type and mutated IRF6 using SBP-HIS tandem pull-down assays. These assays were also conducted with HOK cells stably expressing either SBP-HIS-tagged IRF6- c.748C>T:p.R250X or wild-type IRF6. Both cell types expressed the SBP-HIS-tagged protein, albeit the molecular weight of IRF6-c.748C>T:p.R250X was found to be lesser, indicating that mutation led to a truncating of the protein (Figure 2C).

Subsequent mass spectrometry analysis illuminated stark differences in protein-binding affinities. Of the 374 proteins that exhibited differential binding capacities to the mutant IRF6 in the mutant IRF6-expressing HOK cells compared to the wild-type IRF6, a refined list of 116 proteins was established post-exclusion of non-specific binding entities and proteins identified with less than two unique peptides. Detailed examination revealed that 52 proteins had diminished binding affinities to the mutant IRF6, whereas 27 exhibited enhanced binding in the context of the mutated IRF6. In the subset of proteins with reduced binding affinity, N-α-acetyltransferase 10 (NAA10), small ribonucleic acid polypeptide N (SNRPN), and ribosome assembly protein 1 (NAP1L1) were notably identified (Table 3). Additionally, further analysis revealed four phenotypically pertinent proteins—methylenetetrahydrofolate dehydrogenase, cyclase and formyltetrahydrofolate synthase 1 (MTHFD1), lysine-specific histone demethylase 1A (KDM1A), histone deacetylase 1 (HDAC1), and H2A.Z mutation histone 1 (H2AFZ)—whose binding capacity to mutant IRF6 was significantly lower compared to wild-type IRF6 (Table 4). These findings underscored the intricate molecular landscapes dictated by the IRF6 mutations and their consequent impact on protein-protein interactions.

To validate the influence of the newly discovered mutant loci on the functionality of the IRF6 gene, the HOK cells infected with the lentivirus loaded with wild-type IRF6 and mutant IRF6 underwent RNA sequencing was conducted on HOK cells. Our findings revealed that there was a reduced number of DEGs in the HOK cells overexpressing the mutant IRF6 compared to the HOK cells that overexpressed wild-type IRF6 (Figure 3A). A profound transcriptomic shift was observed in the HOK cells harboring the mutant IRF6. Specifically, our analysis revealed 27 genes with increased expression and 38 with diminished expression in cells overexpressing the mutant IRF6 gene (Figure 3B). This signified potential alterations in cellular signaling dynamics that were attributed to mutations in IRF6. Through comprehensive Gene Ontology (GO) analysis, we delineated that the mutated IRF6 primarily impacted the gene expressions integral to cell adhesion and epithelial cell migration, among other cellular functions (Figure 3C).

By comparing the RNA-seq data between the mutant and wild-type groups, we observed a significant decrease in the expression of TGFβ2-AS1. Given that this lncRNA is involved in the TGFβ signaling pathway, we used qPCR to validate whether reduced expression of TGFβ2-AS1 in HOK cells stably expressing either wild-type or mutant IRF6. Our findings revealed a notable reduction in the expression levels of TGFβ2-AS1 in the cells expressing the mutant. Furthermore, there was a significant reduction in the expression levels of its downstream effectors, CTGF and TGFβ2, compared to the HOK cells overexpressing IRF6, yet there were no marked changes relative to the control group (Figure 4A).

To investigate the impact of IRF6-WT and IRF6-MU on the regulation of signaling pathways, we conducted Western blot analysis to assess their effects on the phosphorylation levels of SMAD2/3 (Figure 4B). In this experiment, HOK cells were transfected with an empty pCDH-SBP-HIS₈ vector plasmid (control), and pCDH-IRF6-WT-flag-puro and pCDH-IRF6-MU-flag-puro plasmids (experimental groups) for comparison. Additionally, we co-transfected HOK cells with the pEGFP plasmid as an indicator to validate transfection efficiency. The results of Western blot revealed that IRF6 plays a role in enhancing the expression and phosphorylation of SMAD3 while also promoting the phosphorylation of SMAD2, effectively activating the TGFβ signaling pathway. Conversely, the p. R250X mutation in IRF6 was found to inhibit the expression of SMAD3 as well as the phosphorylation of both SMAD3 and SMAD2, thereby suppressing the activity of the signaling pathway. Similarly, HOK cells transfected with the pGreen-coGFP-puro vector plasmid served as a control group for comparison with cells transfected with the pGreen-coGFP-TGFβ2-AS1-puro plasmid. We observed that both the total and phosphorylated levels of SMAD3 and SMAD2 were higher in the cells overexpressing TGFβ2-AS1 compared to the control group and were consistent with that of the corresponding levels in the cells overexpressing IRF6-p.R250X. This indicated that TGFβ2-AS1 is a key component of the TGFβ pathway by promoting the expression and phosphorylation of SMAD3 and SMAD2 within the pathway.

Consequently, we proposed that the newly identified mutation in IRF6 within the VWS lineage impaired its capacity to interact with proteins associated with histone modification. This impairment resulted in decreased TGFβ2-AS1 levels and inhibited the phosphorylation of SMAD3, which in turn critically regulated the activity of the TGFβ signaling pathway during embryogenesis. These molecular disruptions culminated in the pathogenesis observed in VWS (Figure 5).